Myoblast Transfer Research Articles

Stem cell route to neuromuscular therapies.

As applied to skeletal muscle, stem cell therapy is a reincarnation of myoblast transfer therapy that has resulted from recent advances in the cell biology of skeletal muscle. Both strategies envisage the reconstruction of damaged muscle from its precursors, but stem cell therapy employs precursors that are earlier in the developmental hierarchy. It is founded on demonstrations of apparently multipotential cells in a wide variety of tissues that can assume, among others, a myogenic phenotype. The main demonstrated advantage of such cells is that they are capable of colonizing many tissues, including skeletal and cardiac muscle via the blood vascular system, thereby providing the potential for a body-wide distribution of myogenic progenitors. From a practical viewpoint, the chief disadvantage is that such colonization has been many orders of magnitude too inefficient to be useful. Proposals for overcoming this drawback are the subject of much speculation but, so far, relatively little experimentation. This review attempts to give some perspective to the status of the stem cell as a therapeutic instrument for neuromuscular disease and to identify issues that need to be addressed for application of this technology.

Apoptosis coincident with the differentiation of skeletal myoblasts is delayed by caspase 3 inhibition and abrogated by MEK-independent constitutive Ras signaling

We demonstrate that during 23A2 skeletal myoblast differentiation, between 30-35% of the population apoptose. Both differentiation and apoptosis are controlled by the variables of cell density and time and these variables are inversely related. In response to conditions that permit both differentiation and apoptosis of parental 23A2 myoblasts, myoblasts rendered differentiation-defective by constitutive Ras signaling (A2:H-Ras myoblasts) do not apoptose. This is not merely a consequence of their differentiation-defective phenotype since myoblasts rendered differentiation-defective by expression of E1A (A2:E1A myoblasts) still apoptose. Although signaling through MEK is important to the survival of proliferating parental 23A2 myoblasts, constitutive signaling through MEK is not responsible for the survival of A2:H-Ras myoblasts. Finally, we demonstrate that caspase 3 is activated and that pharmacological inhibition of caspase 3 activity delays apoptosis without affecting differentiation. Abrogating apoptosis without affecting differentiation could be a useful approach to improve the efficacy of myoblast transfer in the treatment of muscular dystrophies.

SURGICAL TREATMENTS FOR HEART FAILURE

The surgical options for CHF are a part of a larger paradigm shift in management. Viable and effective surgical options other than cardiac transplant and ventricular assist devices clearly exist and are applicable to a large portion of patients with CHF. These surgical therapies are of acceptable risk before decompensated CHF develops. The rapidly evolving therapies for altering LV remodeling, which underlies CHF progression, are an exciting area that may be joined in the future by molecular advances in myoblast transfer and gene therapy. These therapies are the basis of the discipline of CHF surgery within cardiovascular surgery.

Absence of MyoD Increases Donor Myoblast Migration into Host Muscle

Donor myoblast migration is a major limiting factor in the success of myoblast transfer therapy, a potential treatment for Duchenne muscular dystrophy. A possible strategy to promote the migration of donor myoblasts into host muscle is to enhance their proliferation and delay their fusion, two properties that are major characteristics of myoblasts in regenerating skeletal muscle in MyoD null (−/−) mice. Here we investigate whether the migration of MyoD (−/−) donor myoblasts into host muscle is enhanced in vivo. Sliced muscle grafts from male MyoD (−/−) or normal control (Balb/c) mice were transplanted into the muscles of female normal (Balb/c) host mice. Muscles were sampled at 1, 3, and 12 weeks after grafting, and the fate of male donor myoblasts within female host muscles determined by in situ hybridization with the mouse Y-chromosome-specific Y-1 probe. MyoD (−/−) donor myoblasts migrated into host muscle continuously over 1, 3, and 12 weeks after grafting, in contrast with Balb/c donor myoblasts, whose overall numbers and migratory distances did not increase significantly after 1 week. These results strongly support a role for elevated donor myoblast proliferation and/or their delayed fusion in enhancing migration into host muscle in vivo, and endorse the use of either genetically engineered donor myoblasts, or the administration of exogenous myoblast mitogens to improve donor myoblast migration in myoblast transfer therapy.

Complement and myoblast transfer therapy: donor myoblast survival is enhanced following depletion of host complement C3 using cobra venom factor, but not in the absence of C5.

Myoblast transfer therapy (MTT) is a potential cell therapy for myopathies such as Duchenne Muscular Dystrophy and involves the injection of cultured muscle precursor cells ('myoblasts') isolated from normal donor skeletal muscles into dystrophic host muscle. The failure of donor myoblast survival following MTT is widely accepted as being due to the immune response of the host. The role of complement as one possible mechanism for the initial, very rapid death of myoblasts following MTT was investigated. Donor male myoblasts were injected into the tibialis anterior (TA) muscles of female host mice that were: (i) untreated; (ii) depleted of C3 complement (24 h prior to MTT) using cobra venom factor (CVF); and/or (iii) deficient in C5 complement. Quantification of surviving male donor myoblast DNA was performed using the Y-chromosome specific (Y1) probe on slot blots for samples taken at 0 h, 1 h, 24 h, 1 week and 3 weeks after MTT. Peripheral depletion of C3 was confirmed using double immunodiffusion, and local depletion of C3 in host TA muscles was confirmed by immunostaining of muscle samples. Cobra venom factor treatment significantly increased the initial survival of donor myoblasts, but there was a marked decline in myoblast numbers after 1 h and little long-term benefit by 3 weeks. Strain specific variation in the immediate survival of donor male myoblasts following MTT in untreated C57BL/10Sn, DBA-1 and DBA-2 (C5-deficient) female hosts was observed. Cobra venom factor depletion of C3 increased initial donor male myoblast survival (approximately twofold at 0 h) in C57BL/10Sn and DBA-1 host mice and approximately threefold in DBA-2 hosts at 0 h and 1 h after MTT. The rapid and extensive number (approximately 90%) of donor male myoblasts in untreated DBA-2 mice (that lack C5) indicates that activation of the membrane attack complex (MAC) plays no role in this massive initial cell death. The observation that myoblast survival was increased in all mice treated with CVF suggests that CVF may indirectly enhance donor myoblast survival by a mechanism possibly involving activated C3 fragments.

Cause of progression in Duchenne muscular dystrophy: impaired differentiation more probable than replicative aging.

Replicative aging of myogenic cells (satellite cells) owing to enhanced myofiber turnover is a common explanation of the progression of Duchenne muscular dystrophy (DMD). This hypothesis has been reexamined recently by telomere length measurements in dystrophic tissue. We evaluate the controversial results of these studies. We also review a large body of in vitro, animal (mdx), and patient data which indicate that impaired differentiation, but not replicative aging, is the leading cause of progression in DMD. We recommend in vivo investigations of cell kinetics in DMD muscle, as well as telomere length and telomerase analyses of DMD satellite cells in vitro for a definite judgement of the replicative aging hypothesis. Analogous investigations were helpful in AIDS research where replicative aging was embraced as a simple explanation of the paradigmatic CD4 lymphocyte decline but had to be rejected in favour of more complex models of disturbed lymphocyte homeostasis and regeneration. The question of replicative aging versus impaired differentiation is relevant for the understanding of therapeutic failures and the design of new strategies. Impaired differentiation is compatible with the failure of myoblast transfer in DMD and calls for further studies on the myofiber environment. Replicative aging, on the other hand, could possibly be treated by telomerase gene delivery.

Efficient non-viral DNA-mediated gene transfer to human primary myoblasts using electroporation

Gene transfer of human primary myoblasts with various non-viral methods has been hampered by low yield of transfection. We report here an efficient, simple and reproducible non-viral DNA-mediated gene transfer procedure for transfecting human myoblasts. We found that electroporation promotes a highly efficient DNA uptake by human primary cultures of myogenic cells. Under optimal conditions, 60-70% of human myoblasts transfected with the enhanced green fluorescent gene expressed the enhanced green fluorescent protein. Electroporated myoblasts behaved normally as judged by their ability to synthesize and express developmentally regulated proteins and to undergo terminal differentiation, i.e. to fuse and form myotubes. We showed, in addition, that a subpopulation of cultured human myoblasts with self-renewing properties and equivalent to native muscle satellite cells were as efficiently transfected by electroporation as proliferating myoblasts. Thus, the development of gene therapies based on the engineering and transplantation of human myoblasts may greatly benefit from gene transfer by electroporation.

Leukemia inhibitory factor enhances regeneration in skeletal muscles after myoblast transplantation.

Cell-based therapies, such as myoblast transfer therapy, are likely to become an integral part of any approach to treat myopathies such as Duchenne muscular dystrophy. Previous studies have shown that an increased level of regeneration in the host muscle enhances incorporation of donor myoblasts. Leukemia inhibitory factor (LIF) increases the number of dystrophic fibers expressing dystrophin after myoblast transplantation and enhances regeneration in injured and diseased muscle. Morphometric analysis was used to investigate whether an increased level of regeneration is induced by LIF after myoblast transplantation. We found that, in muscles treated with LIF, the number of fibers undergoing regeneration was increased. The increased incorporation of donor myoblasts and thus dystrophin expression induced by LIF may be due, at least in part, to an increased level of regeneration of dystrophic muscle.

Cell transplantation--a potential therapy for cardiac repair in the future?

Adult human myocardium cannot regenerate because cardiac muscle cells do not reenter the cell cycle. Myoblasts, cardiomyocytes, and stem cell-derived cardiomyocytes have been transplanted in experimental settings to replace lost myocardial tissue. The purpose of this paper is to review the experimental data about cell transfer onto myocardium and to highlight the advantages of the particular cell types used. Myoblasts or satellite cells are precursor cells attached to skeletal muscle fibers. The transfer of these cells onto damaged myocardium was demonstrated successfully in several animal models. Because myoblasts can be expanded in culture, a large number of cells can be obtained from only a small skeletal muscle specimen. These cells could be delivered locally by injection within a damaged myocardial area or by coronary infusion. However, only one group has been able to show an improvement in myocardial function after myoblast transfer. The second type of cells used experimentally for cell transfer were fetal cardiomyocytes. Fetal cardiomyocytes retain the ability to divide and therefore can be expanded in culture. The cells were integrated into the myocardial tissue, differentiated into normal cardiac muscle cells, and formed intercellular connections with host myocardial cells. The transplantation of these cells onto cryo-injured myocardium resulted in improved cardiac function in several animal models. Stem cell-derived cardiomyocytes can be selected from embryonic stem cells. These cells still divide and differentiate into different cardiac muscle cells (atrial, ventricular, and pacemaker). After transplantation into damaged myocardium in mice, they formed stable grafts and survived for at least 7 weeks. The selection of these cells has to be performed with care to prevent teratoma formation originating from single undifferentiated cells attached to the transferred cells. Recent experimental studies revealed the ability of bone marrow stem cells to differentiate into cardiomyocytes. These cells were transplanted into damaged myocardium via coronary perfusion. They survived for at least 4 weeks and showed differentiation toward cardiac muscle cells. The functional benefit of bone marrow stem cells, however, has not been clearly demonstrated, and there is a possibility of tumor formations originating from these cells. Myoblasts and bone marrow stem cell-derived cardiomyocytes would permit autologous cell transfer onto the myocardium. These cells can be easily obtained and expanded in culture. Gene transfer is also possible, and there are no ethical proscriptions against an autologous cell transfer. However, the integration and final differentiation of these cells in the heart tissue is not clear yet. Fetal cardiomyocytes, on the other hand, are integrated in the myocardial tissue, improve cardiac function, and can be expanded in culture. Their transfer would be allogenic, making immunosuppression necessary. Stem cell-derived cardiomyocytes could be used to replace all 3 types of cardiac muscle cells, and they can be expanded in culture. The possibility of teratoma formation makes a 100%- selection mandatory. At present, ethical concerns against working with human embryonic stem cells are a factor to be considered. Cell transfer therapy has been shown to improve myocardial function in animal experiments. This finding indicates that a reduced myocardial function can be improved by cell transfer therapy. Stem cell-derived cardiomyocytes in particular, either of embryonic or bone marrow cell origin, would allow for selective replacement of pacemaker cells or atrial or ventricular cardiomyocytes.

Problems and solutions in myoblast transfer therapy.

Duchenne muscular dystrophy is a severe X-linked neuromuscular disease that affects approximately 1/3500 live male births in every human population, and is caused by a mutation in the gene that encodes the muscle protein dystrophin. The characterization and cloning of the dystrophin gene in 1987 was a major breakthrough and it was considered that simple replacement of the dystrophin gene would ameliorate the severe and progressive skeletal muscle wasting characteristic of Duchenne muscular dystrophy. After 20 years, attempts at replacing the dystrophin gene either experimentally or clinically have met with little success, but there have been many significant advances in understanding the factors that limit the delivery of a normal dystrophin gene into dystrophic host muscle. This review addresses the host immune response and donor myoblast changes underlying some of the major problems associated with myoblast-mediated dystrophin replacement, presents potential solutions, and outlines other novel therapeutic approaches.

The current status of myoblast transfer.

As a means of correcting the defect in the muscles of Duchenne muscular dystrophy (DMD) patients, myoblast transplantation has the advantage of utilizing normal biological mechanisms of repair of this tissue. Thus, it has the potential not only for correcting or complementing the genetic defect, but also for restoring the structure and function of pathologically damaged muscle tissue. However, it is also subject to many of the problems of other genetic therapies for muscle tissue, especially the problem of dispersing the therapeutic agent throughout the muscle mass and that of immune rejection of the genetically corrected tissue. In this article, these problems are discussed. In addition the history of cell transplantation therapy for DMD is presented as an example of the practical difficulties that arise from over eager application to human trials of preliminary data from animal experiments. Recent demonstrations that stem or early precursor cells from a muscle and from the bone marrow are able to disperse to sites of muscle damage via the blood vascular system and to participate in muscle repair raise the hope that this mode of therapy may be applicable to muscular dystrophies.

AM424: history of a novel drug candidate.

1. Leukaemia inhibitory factor (LIF) is a 180 amino acid single-chain protein, named after its effect on haematopoietic cells. Leukaemia inhibitory factor belongs to a group of cytokines that includes ciliary neurotrophic factor, interleukin (IL)-6, IL-11, cardiotrophin-1 and oncostatin M. All group members use the gp130 signal transducing subunit for intracellular signalling, but show differences in biological effect. 2. Research over the past 6-8 years has shown LIF to have potent neuromuscular activity. In vitro and in vivo studies on axotomy and nerve crush models demonstrate a powerful effect of LIF in enhancing the survival of both motor and sensory neurons, while reducing denervation-induced muscle atrophy. In models of both axotomy induced neuronal death and in the wobbler mouse, LIF is active at doses as low as 1 microgram/kg delivered systemically. 3. In muscle, LIF will increase the rate of muscle regeneration in vivo when applied exogenously after injury and will stimulate intrinsic muscle repair following its targeted release to dystrophic muscle in the mdx mouse. Leukaemia inhibitory factor may also have a role as an adjunct to myoblast transfer therapy, with studies showing that the transplantation of genetically competent myoblasts into mdx mouse muscle is enhanced when cells are injected with LIF. 4. Distribution and pharmacokinetic studies have been conducted in primates with doses of 20 micrograms/kg recombinant human LIF given subcutaneously over 2 weeks tolerated without major side effects. 5. A pharmaceutical form of recombinant human LIF (AM424; AMRAD Operations, Richmond, Victoria, Australia) entered human clinical trials during 1997 and a phase I clinical trial in healthy volunteers has been completed. A phase I repeat dose study has also been completed in cancer patients undergoing chemotherapy. The primary indication for a phase II study is the treatment of chemotherapy induced peripheral neuropathy. Other potential indications include muscle wasting diseases, acute nerve trauma and motor neuron disease. 6. The role of LIF in modulating nerve loss should make it an ideal candidate for the treatment of a number of neurological conditions. The phase I study represents the first trial in a programme for the clinical development of AM424.

Thymic myoid cells as a source of cells for myoblast transfer.

Transplantation of disaggregated myoblasts from normal donor to the muscles of a diseased host, or reimplantation of genetically modified host myoblasts, has been suggested as a possible route to therapy for inherited myopathies such as Duchenne muscular dystrophy, or to supply missing proteins that are required systemically in diseases such as hemophilia. With two exceptions, studies of myoblast transfer in the mouse have involved transplantation of donor myoblasts isolated from adult or neonatal skeletal muscle satellite cells. In this study we present evidence that thymic myoid cells are capable of participating in the regeneration of postnatal skeletal muscle, resulting in the expression of donor-derived proteins such as dystrophin and retrovirally encoded proteins such as beta-galactosidase within host muscles. This leads us to conclude that thymic myoid cells may provide an alternative to myoblasts derived from skeletal muscle as a source of myogenic cells for myoblast transfer.

Why do cultured transplanted myoblasts die in vivo? DNA quantification shows enhanced survival of donor male myoblasts in host mice depleted of CD4+ and CD8+ cells or Nk1.1+ cells.

Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a 32P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10-18% of the 2.5 x 10(5) cells originally injected (designated 100%). This declined further over 1 week to approximately 1-4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early "P3" or late "P15-20" passage) made no difference to this result. Modulation of the host response by CD4+/CD8+ -depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.

Deflazacort increases laminin expression and myogenic repair, and induces early persistent functional gain in mdx mouse muscular dystrophy.

Deflazacort slows the progress of Duchenne muscular dystrophy (DMD) with fewer side effects than prednisone. In mdx mice, deflazacort treatment augments repair and growth of new muscle fibers. We tested the hypothesis that deflazacort improves muscle function and promotes repair by increasing myogenic cell proliferation and fiber differentiation. mdx mice (3.5 weeks old) were treated with deflazacort (1.2 mg/kg) or vehicle for 4 weeks. Forelimb grip strength was measured. After 4 weeks, the right tibialis anterior muscle (TA) was crush injured to induce synchronous regeneration. DNA was labeled using different markers 24 and 2 h before collecting tissues 4 days after injury. The expression of creatine kinase (CK) isoforms, laminin-2 (merosin) mRNA and protein, and proliferation by myogenic cells were measured and satellite cells were identified by immunolocalization of c-met receptor. Peak grip strength increased 15% within 10 days of treatment, and was maintained up to 6 weeks after the end of treatment in a second experiment. Expression of CK MM in the regenerating TA rose from 46% to 55% of total CK activity after deflazacort treatment. Satellite cells were more numerous and appeared earlier on new fibers, in concert with a threefold increase in proliferation by myogenin+ (but not MyoD+) myoblasts. alpha2-Laminin mRNA expression and protein increased 1.3-5.5-fold relative to MM CK in regenerating and dystrophic TA, respectively. These studies support the hypothesis that deflazacort promotes functional gains, myogenic differentiation, myoblast fusion, and laminin expression in regenerating dystrophic muscle. The potential to augment precursor specification, strength, and possible membrane stability may be useful in directing long-term benefits for DMD patients and short-term amplification of precursors prior to myoblast transfer.

Enhancement of adult muscle regeneration by primary myoblast transplantation.

Extensor digitorum longus muscles (EDL) of SCID mice were induced to undergo degeneration-regeneration subsequent to orthotopic, whole-muscle transplantation. Two days after transplantation some of these muscles received injections of primary myoblasts derived from EDL muscles of transgenic mice, which express nuclear localizing beta-galactosidase under the control of the myosin light-chain 3F promoter and enhancer. Nine weeks after transplantation, regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received no further treatment and to unoperated EDL muscles in order to determine the effect of myoblast transfer on muscle regeneration. Many myofibers containing donor derived myonuclei could be identified in the regenerated muscles that had received exogenous myoblasts. The mass of the muscles subjected to transplantation only was significantly less (31% less) than that of unoperated muscles. The addition of exogenous myoblasts to the regenerating EDL resulted in a muscle mass similar to that of unoperated muscles. The absolute twitch and tetanic tensions and specific twitch and tetanic tensions of transplant-only muscles were 28%, 36%, 32%, and 41%, respectively, of those of unoperated muscles. Myoblast transfer increased the absolute twitch and tetanic tensions of the regenerated muscles by 65% and 74%, respectively, and their specific twitch and tetanic tensions were increased by 41% and 48%, respectively. These data suggest a possible role for the addition of exogenous, primary myoblasts in the treatment of traumatized and/or diseased muscles that are characterized by myofiber loss.

Exposure to tissue culture conditions can adversely affect myoblast behavior in vivo in whole muscle grafts: implications for myoblast transfer therapy.

The effects of tissue culture conditions on the viability of myoblasts in whole muscles transplanted in vivo were investigated. Whole male (SJL/J) donor muscles were exposed to various tissue culture reagents and proteolytic enzymes, and allografted into female (SJL/J) host mice. Desmin immunohistochemistry was used to assess the numbers of myogenic cells (as an index of myoblast viability and the extent of regeneration) in tissue sections of whole-muscle grafts sampled on days 7 and 14. DNA quantitation with a Y-chromosome-specific probe was used to determine the total Y-1 sequence DNA (as an index of myoblast survival and proliferation) in whole-muscle grafts sampled on days 1, 3, and 7. In grafts exposed to serum-free medium, there was a delay in myoblast fusion at 7 days that was recovered by 14 days, but exposure to serum (10% or 20%) had a prolonged adverse effect on myotube formation at 14 days. DNA quantitation demonstrated that either serum-free culture medium or 10% serum enhanced the number of male cells within whole-muscle grafts at 7 days. Proteolytic digestion (even for 5 min) of whole muscles prior to grafting was extremely detrimental to myoblast survival and viability at 7 and 14 days. The unexpected finding of adverse effects of tissue culture conditions on the regeneration of whole-muscle grafts in vivo appears to parallel the major problem of the rapid death of isolated cultured donor myoblasts after injection in myoblast transfer therapy. The use of whole-muscle grafts provides an alternative and sensitive model to analyze the crucial effects of various tissue culture components on the subsequent survival and proliferation of myogenic cells in vivo.

Enhanced migration and fusion of donor myoblasts in dystrophic and normal host muscle

Sliced male C57Bl/10Sn (H2-b) donor muscles were grafted into the female histocompatible muscles of untreated, FK506-treated, and T-cell depleted (with or without thymic tolerization) dystrophic (mdx; H2-b) and normal (C57Bl/10Sn; H2-b) hosts, and also into histoincompatible normal (Balb/c; H2-d) hosts. The fate of male donor nuclei was monitored on tissue sections by in situ hybridization with a Y-chromosome specific probe. The results demonstrate that the dystrophic environment is more conducive than normal muscle to donor myoblast migration, with the distance moved being threefold greater at 12 weeks in dystrophic hosts. T-cell depletion was significantly more effective than FK506 treatment at enhancing donor myoblast emigration in both histocompatible and histoincompatible hosts at 3 weeks. Furthermore, the effects of T-cell depletion were sustained in histoincompatible hosts at 12 weeks. These data endorse the use of host T-cell depletion as a promising long-term strategy to improve myoblast transfer therapy (MTT) in the clinical situation. © 2000 John Wiley & Sons, Inc. Muscle Nerve 23: 560–574, 2000.

Immunobiology and the future of myoblast transfer therapy.

Myoblast transfer therapy (MTT) is a cell-mediated gene transfer method aimed at the restoration of normal dystrophin expression in Duchenne muscular dystrophy (DMD). Initial clinical MTT trials were conducted amid much controversy, as they were based on very few animal studies. Unfortunately, the trials were of little therapeutic benefit. As a result, there has been a renaissance of interest in experimental studies in animal models. In MTT, myoblasts are obtained by muscle biopsy from normal, i.e., dystrophin-positive, donors, expanded in culture, and injected directly into the muscles of dystrophic recipients. The major requirement for successful MTT is the survival of injected donor myoblasts in the host environment. However, a vast majority of donor cells fail to survive for more than 1 h after injection, and very few last beyond the first week. This review on the immunological aspects of MTT focuses in particular on the roles of specific components of the host immune response, the effects of tissue culture on donor cells, and strategies under development to circumvent the problem of donor myoblast death after injection in vivo.

Enhanced migration and fusion of donor myoblasts in dystrophic and normal host muscle.

Sliced male C57Bl/10Sn (H2-b) donor muscles were grafted into the female histocompatible muscles of untreated, FK506-treated, and T-cell depleted (with or without thymic tolerization) dystrophic (mdx; H2-b) and normal (C57Bl/10Sn; H2-b) hosts, and also into histoincompatible normal (Balb/c; H2-d) hosts. The fate of male donor nuclei was monitored on tissue sections by in situ hybridization with a Y-chromosome specific probe. The results demonstrate that the dystrophic environment is more conducive than normal muscle to donor myoblast migration, with the distance moved being threefold greater at 12 weeks in dystrophic hosts. T-cell depletion was significantly more effective than FK506 treatment at enhancing donor myoblast emigration in both histocompatible and histoincompatible hosts at 3 weeks. Furthermore, the effects of T-cell depletion were sustained in histoincompatible hosts at 12 weeks. These data endorse the use of host T-cell depletion as a promising long-term strategy to improve myoblast transfer therapy (MTT) in the clinical situation.